Injection of lipopolysaccharide induces the migration of splenic neutrophils to the T cell area of the white pulp: role of CD14 and CXC chemokines.

There is increasing evidence that neutrophils are involved in the regulation of adaptive immunity. We therefore tested whether these cells may colocalize with T lymphocytes in lymphoid organs. Our results demonstrate that administration of the microbial product LPS induces the migration of neutrophils in the spleen from the red pulp and the marginal zone to the area of the white pulp where T cells reside. This movement is CD14-dependent, whereas the recruitment of neutrophils in the peritoneal cavity is increased in the absence of CD14. Our data further suggest the involvement of the chemokine MIP-2 and keratinocyte-derived chemokine and their receptor CXCR2. We conclude that neutrophils may interact with naïve T cells upon infection/inflammation and that the migration of neutrophils in the lymphoid organs and in the periphery is regulated differently by a signal transduced by CD14.

Normal thyroid structure and function in rhophilin 2-deficient mice.

Rhophilin 2 is a Rho GTPase binding protein initially isolated by differential screening of a chronically thyrotropin (TSH)-stimulated dog thyroid cDNA library. In thyroid cell culture, expression of rhophilin 2 mRNA and protein is enhanced following TSH stimulation of the cyclic AMP (cAMP) transduction cascade. Yeast two-hybrid screening and coimmunoprecipitation have revealed that the GTP-bound form of RhoB and components of the cytoskeleton are protein partners of rhophilin 2. These results led us to suggest that rhophilin 2 could play an important role downstream of RhoB in the control of endocytosis during the thyroid secretory process which follows stimulation of the TSH/cAMP pathway. To validate this hypothesis, we generated rhophilin 2-deficient mice and analyzed their thyroid structure and function. Mice lacking rhophilin 2 develop normally, have normal life spans, and are fertile. They have no visible goiter and no obvious clinical signs of hyper- or hypothyroidism. The morphology of thyroid cells and follicles in these mice were normal, as were the different biological tests performed to investigate thyroid function. Our results indicate that rhophilin 2 does not play an essential role in thyroid physiology.

STAT5 is an ambivalent regulator of neutrophil homeostasis.

BACKGROUND: Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that in STAT5-/- mice, liver endothelial cells (LECs) autonomously secrete high amounts of G-CSF, allowing myeloid progenitors to overcompensate for their intrinsic survival defect. However, when injected with pro-inflammatory cytokines, mutant mice cannot further increase neutrophil production, display a severe deficiency in peripheral neutrophil survival, and are therefore unable to maintain neutrophil homeostasis. In wild-type mice, inflammatory stimulation induces rapid STAT5 degradation in LECs, G-CSF production by LECs and other cell types, and then sustained mobilization and expansion of long-lived neutrophils. CONCLUSION: We conclude that STAT5 is an ambivalent factor. In cells of the granulocytic lineage, it exerts an antiapoptotic function that is required for maintenance of neutrophil homeostasis, especially during the inflammatory response. In LECs, STAT5 negatively regulates granulopoiesis by directly or indirectly repressing G-CSF expression. Removal of this STAT5-imposed brake contributes to induction of emergency granulopoiesis.

TLR4 and Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta, but not MyD88, regulate Escherichia coli-induced dendritic cell maturation and apoptosis in vivo.

Dendritic cells (DC) are short-lived, professional APCs that play a central role in the generation of adaptive immune responses. Induction of efficient immune responses is dependent on how long DCs survive in the host. Therefore, the regulation of DC apoptosis in vivo during infection remains an important question that requires further investigation. The impact of Escherichia coli bacteremia on DCs has never been analyzed. We show here that i.v. or i.p. administration of live or heat-killed E. coli in mice induces splenic DC migration, maturation, and apoptosis. We further characterize which TLR and Toll-IL-1R (TIR)-containing adaptor molecules regulate these processes in vivo. In this model, DC maturation is impaired in TLR2(-/-), TLR4(-/-) and TIR domain-containing adapter-inducing IFN-beta (TRIF)(-/-) mice. In contrast, DC apoptosis is reduced only in TLR4(-/-) and TRIF(-/-) mice. As expected, DC apoptosis induced by the TLR4 ligand LPS is also abolished in these mice. Injection of the TLR9 ligand CpG-oligodeoxynucleotide (synthetic bacterial DNA) induces DC migration and maturation, but only modest DC apoptosis when compared with LPS and E. coli. Together, these results suggest that E. coli bacteremia directly impacts on DC maturation and survival in vivo through a TLR4-TRIF-dependent signaling pathway.

Treatment of experimental asthma by decoy-mediated local inhibition of activator protein-1.

RATIONALE: Asthma is associated with increased expression of a typical array of genes involved in immune and inflammatory responses, including those encoding the prototypic Th2 cytokines interleukin (IL) 4, IL-5, and IL-13. Most of these genes contain binding sites for activator protein-1 (AP-1) within their promoter and are therefore believed to depend on AP-1 for their expression, suggesting that this transcription factor could be of particular importance in asthma pathophysiology. OBJECTIVE: To clarify the role of AP-1 in the effector phase of pulmonary allergy. METHODS: Ovalbumin (OVA)-sensitized mice were intratracheally given decoy oligodeoxyribonucleotides (ODNs) specifically directed to AP-1 or scrambled control ODNs before challenge with aerosolized OVA. Twenty-four hours after the last OVA challenge, airway hyperresponsiveness was measured and allergic airway inflammation was evaluated quantitatively. AP-1 decoys were localized using flow cytometry and immunohistochemistry. AP-1 activity in the lung was assessed using electrophoretic mobility shift assay. MEASUREMENTS AND MAIN RESULTS: Intratracheally delivered AP-1 decoys efficiently targeted airway immune cells, thus precluding AP-1 activation on OVA challenge. Decoy-mediated local inhibition of AP-1 resulted in significant attenuation of all the pathophysiologic features of experimental asthma-namely, eosinophilic airway inflammation, airway hyperresponsiveness, mucous cell hyperplasia, production of allergen-specific immunoglobulins, and synthesis of IL-4, IL-5, and IL-13. Scrambled control ODNs had no detectable effects. CONCLUSIONS: Our results reveal a key role for AP-1 in the effector phase of pulmonary allergy and indicate that specific AP-1 inhibition in the airways may have therapeutic value in the control of established asthma.

T cell-dependent maturation of dendritic cells in response to bacterial superantigens.

Dendritic cells (DC) express a set of germline-encoded transmembrane Toll-like receptors that recognize shared microbial products, such as Escherichia coli LPS, termed pathogen-associated molecular patterns. Analysis of the in vivo response to pathogen-associated molecular patterns has uncovered their ability to induce the migration and the maturation of DC, favoring thus the delivery of Ag and costimulatory signals to naive T cells in vivo. Bacterial superantigens constitute a particular class of pathogen-derived molecules known to induce a potent inflammatory response in vivo, secondary to the activation of a large repertoire of T cells. We demonstrate in this work that Staphylococcal superantigens induce migration and maturation of DC populations in vivo. However, in contrast to LPS, superantigens failed to induce DC maturation in RAG or MHC class II-deficient mice, suggesting that T cell activation was a prerequisite for DC maturation. This conclusion was further supported by the finding that T cell activation induced by 1) mitogenic anti-CD3 mAbs, 2) allo-MHC determinants, or 3) nominal Ag in a TCR-transgenic model induces DC maturation in vivo. These studies also revealed that DC that matured in response to T cell mitogens display, comparatively to LPS, a distinctive phenotype characterized by high expression of the MHC class II, CD40, and CD205 markers, but only moderate (CD86) to minimal (CD80) expression of CD28/CTLA4 ligands. This work demonstrates that activation of a sufficient number of naive T cells in vivo constitutes a novel form of immune danger, functionally linked to DC maturation.

The adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo.

The aim of this study was to test the capacity of the novel adjuvant OM-174, a lipid A analog, to induce the migration and the maturation of murine dendritic cells (DC) in vivo, a step which is considered as the initiation of the adaptive immune response. BALB/c mice were injected intravenously or subcutaneously with OM-174. The spleen and popliteal lymph nodes were harvested, and analyzed for DC localization and phenotype. The data presented here clearly show that, OM-174 induces the migration of DC from the periphery to the T cell areas of lymphoid organs, and their maturation into cells expressing high levels of MHC class II and co-stimulatory molecules, with a potency close to that of Escherichia coli lipopolysaccharide (LPS).

Selective blockade of NF-kappa B activity in airway immune cells inhibits the effector phase of experimental asthma.

Knockout mice studies have revealed that NF-kappaB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-kappaB also plays a role in the effector phase of airway allergy and whether inhibiting NF-kappaB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-kappaB activity in the lungs during OVA challenge. Intratracheal administration of NF-kappaB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-kappaB activity in the airways upon OVA provocation. NF-kappaB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-kappaB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-kappaB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.

Alteration of migration and maturation of dendritic cells and T-cell depletion in the course of experimental Trypanosoma cruzi infection.

Trypanosoma cruzi, the etiologic agent of Chagas disease, induces infection that affects most immunocompetent cells. However, its effect on dendritic cells (DC) is still unknown in vivo. In this report, we show, by immunohistochemical staining, that T. cruzi infection triggers a huge increase in the number of CD11c(+) DC in the spleen of infected mice at Days 14 and 21 post-inoculation (pi). In mice reaching the chronic phase (starting on Day 35 pi), the number of splenic DC (sDC) returned progressively to normal (ending on Day 98 pi). In the spleens of noninfected mice, most of the CD8alpha(+)CD11c(+) and CD8alpha(-)CD11c(+) DC were found in the red pulp and the marginal and T-cell zones. However, starting on Day 14 pi, a progressive decline of CD8alpha(+)CD11c(+) was observed. In addition, sDC expressed low levels of the costimulatory molecule B7.2 at Days 14 and 21 pi, suggesting that they remained immature in the course of the infection. As expected, in lipopolysaccharide-treated and noninfected mice, the expression of B7.2 molecules was sharply up-regulated on sDC that migrated toward the T-cell zone. In contrast, upon lipopolysaccharide stimulation, sDC from T. cruzi-infected mice did not migrate toward the T-cell zone nor did they undergo maturation. Finally, white pulp was severely depleted in both CD4(+) and CD8(+) T cells at the peak of infection. Taken together, these results indicate that profound alterations of migration and maturation of sDC and depletion/redistribution of T cells occur during the acute phase of T. cruzi infection and could be part of another strategy to escape immune surveillance and to persist in the host.

Amastigote load and cell surface phenotype of infected cells from lesions and lymph nodes of susceptible and resistant mice infected with Leishmania major.

Cells of the dendritic cell (DC) lineage, by their unique ability to stimulate naive T cells, may be of crucial importance in the development of protective immune responses to Leishmania parasites. The aim of this study was to compare the impact of L. major infection on DCs in BALB/c (susceptible, developing Th2 responses), C57BL/6 (resistant, developing Th1 responses), and tumor necrosis factor (TNF)(-/-) C57BL/6 mice (susceptible, developing delayed and reduced Th1 responses). We analyzed by immunohistochemistry the phenotype of infected cells in vivo. Granulocytes (GR1(+)) and macrophages (CD11b(+)) appear as the mainly infected cells in primary lesions. In contrast, cells expressing CD11c, a DC specific marker, are the most frequently infected cells in draining lymph nodes of all mice tested. These infected CD11c(+) cells harbored a particular morphology and cell surface phenotype in infected C57BL/6 and BALB/c mice. CD11c(+) infected cells from C57BL/6 and TNF(-/-) C57BL/6 mice displayed a weak parasitic load and a dendritic morphology and frequently expressed CD11b or F4/80 myeloid differentiation markers. In contrast, some CD11c(+) infected cells from BALB/c mice were multinucleated giant cells. Giant cells presented a dramatic accumulation of parasites and differentiation markers were not detectable at their surface. In all mice, lymph node CD11c(+) infected cells expressed a low major histocompatibility complex II level and no detectable CD86 expression. Our results suggest that infected CD11c(+) DC-like cells might constitute a reservoir of parasites in lymph nodes.

Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) is phosphorylated by Ins(1,4,5)P(3) 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). The physiological function of Ins(1,3,4,5)P(4) is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P(3) 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P(3) amounts or calcium mobilization in Itpkb(-/-) thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P(4) define an essential signaling pathway for T cell precursor responsiveness and development.